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  • Title: Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites.
    Author: Osiecki-Newman K, Fabbro D, Legler G, Desnick RJ, Grabowski GA.
    Journal: Biochim Biophys Acta; 1987 Sep 02; 915(1):87-100. PubMed ID: 2956992.
    Abstract:
    Comparative studies with lipoidal inhibitors and alternative substrates were conducted to investigate the properties of the active site of human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from normal placenta and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the normal enzyme, the inhibitory potencies of series of alkyl(Cn; n = 0-18)amines, alkyl beta-glucosides and alkyl-1-deoxynojirimycins were a biphasic function of increasing chain length: i.e., large decreases in Ki,app or IC50 were found only with n greater than 4 and limiting values were approached with n = 12-14. This biphasic function of alkyl chain length was observed in the presence or absence of detergents and/or negatively charged lipids. In the presence of Triton X-100 concentrations greater than the critical micellar concentration, the relative (to deoxynojirimycin) inhibitory potencies of the N-Cn-deoxynojirimycins (n greater than 4) were decreased about 3-5-fold, due to an energy requirement to extract the inhibitors from Triton X-100 micelles. The Ki,app or IC50 of N-hexylglucosylsphingosine was inversely related to the Triton X-100 concentration and was not affected by the presence of 'co-glucosidase'. The mutual exclusion of glucon, N-Cn-deoxynojirimycin and sphingosine derivatives from the normal enzyme suggested a shared region for binding in the active site. Increasing the fatty-acid acyl chain length of glucosyl ceramide from 1 to 24 carbons had minor effects on Km,app ( = Kis,app) (8-40 microM), but increased Vmax,app up to 13-fold. With the AJGD enzyme, the inhibitor and alternative substrate findings were similar to those with the normal enzyme, except that Kis,app(AJGD)/Kis,app(normal) = 4 to 11 for the Cn-glycons and sphingosine derivatives. These results indicated that (1) the Ki,app or Km,app values for amphiphilic inhibitors or substrates reflect a balance of binding energies for two hydrophobic subsites within the enzyme's active site and Triton X-100 micelles and (2) the abnormal properties of the AJGD enzyme result from an amino-acid alteration(s) within or near a hydrophilic region which is shared by the glycon-binding site and the two hydrophobic sites of the active site.
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